1. Field of the Invention
The present invention relates to high quantum yield chemiluminescent acridinium compounds with increased light output. Structural features necessary for obtaining increased light emission from acridinium compounds are disclosed herein. Additionally, we also disclose hydrophilic versions of these structures, which not only have increased light output but also have increased water solubility and low nonspecific binding. These compounds because of their enhanced quantum yield and hydrophilic nature, are useful in improving assay sensitivity.
2. Background of the Invention
Chemiluminescent acridinium esters (AEs) are extremely useful labels that have been used extensively in immunoassays and nucleic acid assays. A recent review, Pringle, M. J., Journal of Clinical Ligand Assay vol. 22, pp. 105-122(1999) summarizes past and current developments in this class of chemiluminescent compounds.
McCapra, F. et al., Tetrahedron Lett. vol. 5, pp.3167-3172 (1964) and Rahut et al. J. Org. Chem vol. 301, pp. 3587-3592. (1965) disclosed that chemiluminescence from the esters of acridinium salts could be triggered by alkaline peroxide. Since these seminal studies, interest in acridinium compounds has increased because of their utility as labels. The application of the acridinium ester 9-carboxyphenyl-N-methylacridinium bromide in an immunoassay was disclosed by Simpson, J. S. A. et al., Nature vol. 279, pp. 646-647 (1979). However, this acridinium ester is quite unstable, thereby limiting its commercial utility. This instability arises from hydrolysis of the 9-carboxyphenyl ester linkage between the phenol and the acridinium ring.
Different strategies for increasing the stability of acridinium compounds have been described in the prior art. Law et al., Journal of Bioluminescence and Chemiluminescence, vol. 4, pp. 88-89 (1989) introduced two methyl groups to flank the acridinium ester moiety to stabilize this linkage. The resulting sterically stabilized acridinium ester, DMAE-NHS [2′,6′-dimethyl-4′-(N-succinimidyloxycarbonyl)phenyl 10-methylacridinium-9-carboxylate] was found to have the same light output as an acridinium ester lacking the two methyl groups. The stability of the former compound when conjugated to an immunoglobulin was vastly superior and showed no loss of chemiluminescent activity even after one week at 37° C. at pH 7. In contrast, the unsubstituted acridinium ester only retained 10% of its activity when subjected to the same treatment. U.S. Pat. Nos. 4,918,192 and 5,110,932 describe DMAE and its applications.
The sterically-stabilized acridinium ester, DMAE-NHS has been used commercially in the ACS:180™ immunoanalyzer (Bayer Diagnostics). U.S. Pat. No. 5,656,426 to Law et al. discloses a hydrophilic version of DMAE termed NSP-DMAE-NHS ester. Both DMAE and NSP-DMAE are currently used in Bayer's ACS:180™ and Advia Centaur™ immunoanalyzers. The chemical structures of these compounds and the numbering system of the acridinium ring are illustrated in the following figures:

Because the acridinium ring is symmetrical, C-1 is equivalent to C-8, C-2 is equivalent to C-7, C-3 is equivalent to C-6, and C-4 is equivalent to C-5.
U.S. Pat. No. 6,664,043 B2 to Natrajan et al discloses NSP-DMAE derivatives with hydrophilic modifiers attached to the phenol. The structure of one such compound is illustrated in the above figure. In this compound a diamino hexa(ethylene) glycol (diamino-HEG) moiety is attached to the phenol to increase the aqueous solubility of the acridinium ester. A glutarate moiety was appended to the end of HEG and was converted to the NHS ester to enable labeling of various molecules.
A different class of stable chemiluminescent acridinium compounds has been described by Kinkel et al., Journal of Bioluminescence and Chemiluminescence vol. 4, pp. 136-139 (1989) and Mattingly, Journal of Bioluminescence and Chemiluminescence vol. 6, pp. 107-114 (1991) and U.S. Pat. No. 5,468,646. In this class of compounds, the phenolic ester linkage is replaced by a sulfonamide moiety, which is reported to impart hydrolytic stability without compromising the light output. In acridinium esters, the phenol is the ‘leaving group’ whereas in acridinium sulfonamides, the sulfonamide is the ‘leaving group’ during the chemiluminescent reaction with alkaline peroxide.
Light emission from acridinium compounds is normally triggered by alkaline peroxide. The overall light output, which can also be referred to as the chemiluminescence quantum yield, is a combination of the efficiencies of the chemical reaction leading to the formation of the excited-state acridone and the latter's fluorescence quantum yield.
A number of factors can influence the overall light output of acridinium compounds. The intrinsic chemiluminescence quantum yields of acridinium compounds are markedly affected by their structures. While most studies have focused on the effect of the leaving group on light emission, none have addressed the effect of functional groups on the acridinium ring on chemiluminescence quantum yields although their effects on the wavelengths of light emission have been well documented. See U.S. Pat. No. 6,355,803. Although, the synthesis of an acridinium ester with methoxy groups at C-2 and C-7 of the acridinium ring system has also been disclosed in U.S. Pat. No. 5,521,103, the effect of the two methoxy groups on either the quantum yield or wavelength of light emission of the acridinium ester was not disclosed.